Abstract
β-galactosidase (β-D-galactoside galactohydrolase; EC 3.2.1.23) is usually used for producing lactose-free milk, and converting lactose to the prebiotic galacto-oligosaccharides (GOS). In this study, Lactobacillus leichmannii 313 (LL313), as a food grade lactic acid bacterium, was explored as a novel source of β-galactosidase, together with an assessment of the biochemical characteristics of this enzyme. The crude β-galactosidase from LL313 was purified in three steps: ammonium sulfate fractionation, ultrafiltration, and fast protein liquid chromatography (FPLC); followed by investigating the effect of each purification step with specific enzyme assays and SDS-PAGE. After purification, the effect of temperature (37 to 70°C) on the enzyme activity, as well as thermal inactivation (50 to 65°C) of both crude and purified enzymes were investigated to estimate the thermal stability of β-galactosidase from LL313. The effects of pH (4 to 8), mono/divalent cations (sodium, potassium, calcium, magnesium, manganese), and inhibitors (phenylmethylsulphonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), 2-mercaptoethanol) on β-galactosidase activity were also estimated. Lastly, the ability of LL313 -galactosidase to hydrolyze lactose and produce characterized GOS was also demonstrated with the aid of high-performance liquid chromatography (HPLC).
β-galactosidase was produced by fermentation of LL313 in modified MRS broth, in which LL313 grew well. After fermentation, crude β-galactosidase was produced by lysis of the bacterial cells using an ultrasonic probe. The representative specific enzyme activity of crude β-galactosidase using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate was 417 ± 8 µmol·mg-1·h-1.
Crude β-galactosidase from LL313 was purified successively by ammonium sulfate (60%) fraction, ultrafiltration, and ion-exchange chromatography. The purification -fold and activity yield following these steps were 4.5 and 11%, respectively. Based on SDS-PAGE and native PAGE, the molecular weight of β-galactosidase from LL313 is estimated to be 110 kDa.
The optimal temperature of β-galactosidase activity was 55-60℃. The thermal denaturation of this enzyme, as conducted in glass capillaries, showed that thermal inactivation of crude and purified β-galactosidase at 55-65℃ fitted first-order reaction kinetics well (r2 ≥ 0.90). The activation energy (Ea) of crude and purified β-galactosidase inactivation were estimated to be 390.58 ± 34.94 kJ/mol, and 404.17 ± 46.19 kJ/mol respectively, fitting the Arrhenius relation well (Corrected r2 = 0.984 and 0.987 respectively), showing no significant difference in thermal stability between crude and purified β-galactosidase.
The optimal pH value of β-galactosidase from LL313 was 5.5. In relation to the effect of cations on the enzyme activity, β-galactosidase from LL313 was activated by sodium ions and inhibited by calcium and manganese ions. Magnesium ions also inhibited the enzyme activity but at high concentrations (100 mM), while potassium ions had no significant effect on the enzyme activity. Further, at 37 ℃, pH 7, the activity of LL313 β-galactosidase in 50mM potassium phosphate was three times higher than in 50 mM Tris-HCl buffer, but there was no difference between 50 mM Tris-HCl buffer with and without 50 mM KCl, showing that Tris base could be an inhibitor for LL313 β-galactosidase. Regarding inhibitors, β-galactosidase from LL313 was not significantly inhibited by 1 mM PMSF (8%), was significantly inhibited by 1 mM EDTA (46%), and significantly activated by 2-mercaptoethanol (30%). The Vmax value of β-galactosidase hydrolyzing ONPG was 28.83 ± 0.32 nmol·min-1 in this experiment, and Km value was 2.97 ± 0.32 mM.
The hydrolysis products of the action of β-galactosidase from LL313 on lactose (i.e. glucose, galactose, and oligosaccharides) were detected by liquid chromatography and showed that activity of this enzyme for lactose was 92,105 μmol·h-1.mg-1. Moreover, several types of uncharacterized oligosaccharides were generated, demonstrating the feasibility of using LL313 β-galactosidase in the production of GOS. Overall, this study has identified for the first time, the key biochemical characteristics of β-galactosidase from Lactobacillus leichmannii 313, and also demonstrated that this enzyme can be used to hydrolyze lactose and generate GOS. This finding has the potential to expand the food industry applications of LL313 in the processing of GOS prebiotics.